Figure 3. An RFLP linkage map of the five chromosomes of the Arabidopsis thaliana genome. Map distances are shown in centimorgans. (We arbitrarily assigned a position of zero to the top-most marker on each chromosome.) Markers are designated by a clone number for the random low copy clones, and by a clone name for the known genes. An a or b following the marker name indicates the particular RFLP alleles of the clone used to map the locus. Markers scored in both crosses are indicated by ( t::J ). Markers scored only in the cross between Nd-0 and La-O are indicated by ( - ). The remaining markers were scored only in the cross between Nd-0 and C. Markers placed in an order which is less than 1000 times more likely to have given rise to the data than any alternative order are bracketed to the right of the map distances; the numbers by the brackets are the log10 of likelihood ratio of the order shown to the next most likely alternative order. r n three separate cases (115, 254, and 506), the order of markers could not be resolved, i.e., the likelihood for any of the possible intervals was virtually the same; the approximate positions of these markers are indicated to the left of the other markers.
1
2 3 4 5
302 506-448 0 >2.79 447t
0 an+ o 336+
0 0 262 1.9 456 3.9 217 4.1 429 243 7.3 224t
11.1 8.5-0.02~r,
pAtc4 228 518 15.2 A.CHS2 14.8 322 14.2 A. Ph.Ara.1 15.2 :;;-0.26 551 16.3 317 17.2 -0.0 20.2 210 241 19·9 >2.36 323 21.3 105 22.7 455 25.3>
333 23.2 283 25.0 326 28.5° ·
53 291 + 27.8 219 26.5 220 25.6 255 29.2 er 31.0 226 33.1 235t
36.4 433 36.3 251 40.2 557+
44.8 215 465 47.4> g/-1 48.4 310 51.6 216 49.9 0.18 272l52.3 271 53.4 >/.54 104 53.1 247157.756.5 214 60.3>1.14 201 ~U -o.w 422 62.6 321 249 ap-2 62.1 423 254-402 60.8 281b ~~:~ -0.11> 1.10 62.4 >o.o 335 65.1 pGH1 68.9 -0.0 sAt2105 11.2 253 68.9 409 299 497 73.3 457 74.2 A.At-24 75.0 246 80.1 225
+
79.4 281 a±
64.8>
o.23 339f
66.4 331t
90·0 ~ 1.59 213 87.9 115-460 89.2 )2.11 268 90·5 424 93.3 435t
98.7 280!"'
233 100.7>
0.85 nAt1511a 101.0 =- 259 tz 1°4·3-o.o5 nAt1511b 101.9 · 558 105.3 305 107.1 421 109.0 315 113.0 555t
118.2 >0.17 211 121.3 252 123.4 > 009 453 125.5 . c/v-1 131.8 532 134.7>
0.15 237 136.3 jAt3012 137.3 132 144.4Figure 4. A graph depicting the completeness of the RFLP linkage map as measured by the percent of the genome within a given map distance (eM) of an RFLP marker. To avoid assuming lengths for the ends of the chromosomes, the genome is considered here to be the sum of the RFLP chromosome lengths, where the two ends of each chromosome are defined by the outermost RFLP markers.
-
Q) c~ Q)
a.
80
60
40
20
o+-~-,--~,_~-,--~~~~--~~~-,~
0 2 4 6 8 10 12 14
centimorgans
CONCLUSION
molecular genetics as part of a larger effort to learn about the molecular, physiological, and biochemical processes of higher plants. Using Arabidopsis as a model system, the work described in this thesis examines and provides approaches for increasing our understanding of gene function in plant growth and development. One approach facilitates the study of gene expression and the other allows for the isolation of a wide range of genes. The latter is related to the unusual size and organization of the Arabidopsis genome. In addition, experiments in this thesis add to our knowledge of genetic structure and gene regulation in flowering plants.
The alcohol dehydrogenase (ADH) gene was one of the first genes to be cloned in Arabidopsis and the first to be sequenced. Two general principles concerning Arabidopsis genes were introduced by the cloning and characterization of this gene. The first is that genes can be cloned from an Arabidopsis DNA library using heterologous gene probes.
(For ADH, this reflects the conservation between monocots and dicots which are thought to have diverged 150 million years ago.) Thus genes initially isolated in Arabidopsis will be useful for obtaining the homologous genes in other plants of interest. Second, Arabidopsis genes contain fewer and smaller introns compared with the introns of homologous genes in other experimental plants. In addition, whereas other plants may contain several genes in a gene family, genes in Arabidopsis are present in fewer copies. In general, the dozen or so genes recently isolated in Arabidopsis (mostly using heterologous probes) have upheld these observations made with the ADH gene. The smaller gene size and the reduced number of genes alone cannot account for the relative difference in genome size between Arabidopsis and other higher plants.
DNA sequence analysis showed that the ADH gene contains sequences characteristic of expressed eukaryotic genes in terms of the TAT A box, polyadenylation signal, and intron structure. From the deduced protein sequence, Arabidopsis ADH is highly conserved with ADH in monocots and mammals, but not in fungi or insects. Part of the 5'
DNA sequence homology between the maize ADHl and the Arabidopsis ADH genes, found in Chapter One, has since been discovered to be a binding site for two proteins in maize upon induction of ADHl (Ferl, R.J., and Nick, H.S., J. Bioi. Chem. 262, 7947- 7950, 1987). This sequence is likely to be involved in regulation of ADH gene expression in Arabidopsis. Tissue- and stage-specific ADH activity was identified using histochemical staining. ADH is constitutively expressed in developing embryos, and is inducible in root tips (as in maize) based on tissue staining and native protein gels. Levels of both ADH message and activity were greater in the Bensheim ecotype than in Landsberg erecta.
ADH gene fusions can potentially be used to isolate mutations in trans-acting regulators of gene expression based on counter-selection of ADH activity. Promoters of tissue- or stage- specific genes can be fused to the structural portion of the ADH gene and then the constructs can be transformed into an ADH null background (the present transformation efficiency is sufficient for this approach). After mutagenesis of a transformed line carrying several copies of the introduced DNA, mutants in trans-acting genes can be selected with allyl alcohol. This approach is presently being taken with the seed-specific genes because allyl alcohol selection has been established at the seed level.
The fact that seeds with low levels of ADH can be selectively eliminated, as shown in Chapter Two, suggests that mutations that do not completely abolish expression can be isolated.
Agrobacterium-mediated transformation of Arabidopsis was shown to be effective for assaying complementation of a mutant allele by a genomic clone carrying the wild-type gene. The work presented here is the first demonstration of this in a higher plant. The transferred DNA is stable and inherited in a Mendelian fashion. However, DNA rearrangements and factors such as genomic location of the introduced DNA were found to affect gene expression. Transformation and complementation of a mutant phenotype show that clones can be assayed for complementation in a chromosome walk, and that large pieces of contiguous DNA are transferred by this procedure. Although the compactness of
transformation is presently much too low to make such an approach practical.
Mapping of restriction fragment length polymorphisms (RFLPs) in Arabidopsis has revealed several principles of genetic organization of this plant, and will continue to be valuable for different kinds of analyses. For example, through mapping it was established that random low copy clones in an Arabidopsis genomic DNA library are randomly distributed in the genome. This information concerning the organization of the Arabidopsis genome was not previously known, and it suggests that low copy sequences in a genomic DNA library are likely to be equally represented (which is important for chromosome walking). It also suggests that there are no large genomic regions for which the relation of physical map distance, measured in kilobase pairs, and meiotic map distances, as measured in centiMorgans, is greatly different from the average. To examine the evolution of plant genomes, linkage of RFLP markers in Arabidopsis can be compared with that in other Brassicaceae. RFLPs are also a way to observe DNA sequence variations between different individuals; the three Arabidopsis ecotypes in Chapter Three were found to diverge approximately equally from each other. Using RFLP mapping, cloned genes (with or without an identified function) have been associated with previously isolated and mapped mutations as shown for chalcone synthase and nitrate reductase.
The RFLP map is a powerful tool for isolating a wide range of genes. The distinct advantage of chromosome walking is that one can start with only a mutant phenotype that can be mapped relative to the RFLP map. The range of obtainable genes is not limited to genes that either have been cloned in heterologous systems, are differentially or abundantly expressed, or for which the corresponding protein is isolated or identifiable. Thus, genes not previously cloned, and especially genes that encode rare messages for (presently) unknown functions, can be obtained. Chromosome walking is facilitated in Arabidopsis due to the small genome and lack of dispersed repeats. These features are not known for any other higher plant. The use of the RFLP map for gene cloning is in progress in both